PCR amplification has traditionally been accomplished via a plurality of amplification cycles, with each cycle comprising the step of initial denaturation, annealing, polymerization, and final extension. These cycles are generally conducted in a reaction chamber, which is provided with necessary PCR reagents, including the biological sample containing the target nucleotide sequence (generally DNA, or RNA) a DNA polymerase (e.g., Taq polymerase), nucleoside triphosphates, an RT enzyme, and a first and second primer (comprising a primer pair) that hybridize to the target DNA and flank the sequence of the amplified DNA product (the “amplicon”). A PCR apparatus will typically include means for cycling the temperature of the reaction chamber as required for each step of the amplification cycle, including, e.g., “melting” of double stranded DNA to produce single stranded DNA; annealing of the primers to single stranded DNA templates; and extension of the amplified DNA via polymerase.
The precise conditions used to amplify a specific target DNA sequence can vary according to a number of factors which are within the knowledge of those of ordinary skill in the art. In some embodiments of traditional DNA amplification, denaturation is conducted at between about 90-95° C. for about 10-30 seconds, annealing is conducted at about 45-65° C. for about 10-30 seconds; extension is conducted at about 70-75° C. for about 10-90 seconds; and a final extension is conducted at 72° C. for about 5 minutes. In some embodiments, the reaction mixture comprises genomic DNA, MgCl2 and other physiological salts (e.g., NaCl), PCR buffer, 0.1-1.0 mM dNTPs, 0.04-1.5 μM of each primer, and 0.5-5.0 units of heat stable polymerase (e.g., Taq. polymerase).
Other amplification methods known in the art may also be utilized, including, for example, self-sustained sequence replication (3SR), strand-displacement amplification (SDA); “branched chain” DNA amplification (Chiron Corp.); ligase chain reaction (LCR), QB replicase amplification (QBR), ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), and cycling probe reaction (CPR) (reviewed, e.g., in The Genesis Report, DX; Vol. 3(4), pp. 2-7 (February 1994)).
Real-time PCR typically relies on the use of fluorescent molecules that allow quantification or detection of a PCR product in real time, while other detection/quantification chemistries such as electrochemistry are also applicable.
Fluorescent molecules can be DNA binding dyes such as SYBR Green or fluorescently labeled primers or probes. There are many fluorescent dyes and probe designs available for different applications. The most commonly used DNA-binding dye for real-time PCR is SYBR® Green I, which binds preferentially to double-stranded DNA (dsDNA) versus single stranded DNA. SYBR Green I fluorescence increases up to 1,000-fold when it binds to dsDNA. Therefore, fluorescence signal is proportional to the amount of dsDNA present.
The major drawback of DNA-binding dyes is their lack of specificity, that is, DNA-binding dyes bind to any dsDNA. As a result, the presence of any nonspecific products in a real-time or endpoint PCR reaction will contribute to the overall fluorescence and affect the accuracy of quantification or detection. Furthermore, DNA-binding dyes cannot be used for quantification or detection in multiplex reactions because fluorescence signals from different products cannot be distinguished without the inclusion of a post PCR melting curve analysis to distinguish the formation of different products.
In contrast, primer-based and probe-based detection chemistries ensure that signal is generated only when the product of interest is amplified. The primer or target-specific oligonucleotide probe is typically labeled with a reporter fluorophore, but in most cases, fluorescence is quenched when the specific target is not yet amplified or when not present in the sample. Usually this is accomplished by attaching a quencher molecule to the primer or probe, and devising some mechanism by which the reporter and quencher are separated when the primer or probe binds to its specific target.
The principal primer/probe detection chemistries in use today are as follows:
Hydrolysis (TaqMan) Probe
Hydrolysis assays include a sequence-specific, fluorescently labeled oligonucleotide probe, in addition to the sequence-specific primers. Hydrolysis assays exploit the 5′ exonuclease activity of certain thermostable polymerases, such as Taq or Tth. The hydrolysis probe is labeled with a fluorescent reporter at one end and a quencher at the opposite end, though several variations on this particular design are in common usage. When the probe is intact, fluorscence is quenched due to fluorophore proximity to the quencher. A commonly used fluorescent reporter-quencher pair is fluorescein (FAM), which emits green fluorescence, and Black Hole Quencher 1 dye, although this is just one of many dye/quencher combinations in use.
The amplification reaction includes a combined annealing/extension step during which the probe hybridizes to the target and the dsDNA-specific 5′ to 3′ exonuclease activity of Taq or Tth cleaves the oligonucleotide, separating fluorophore from quencher, resulting in a fluorescence signal that is proportional to the amount of amplified product in the sample. A properly designed Hydrolysis probe can be used in combination with additional probes of similar design to determine sequence variations within the amplified target, i.e. genotype.
Molecular Beacons
Molecular beacons are dye-labeled oligonucleotides (25-40 nt) that form a hairpin structure. The 5′ and 3′ ends have complementary sequences of 5-6 nucleotides that form the stem, while the loop is designed to specifically hybridize to a 15-30 nucleotide section of the target sequence. A fluorescent reporter molecule is attached to one end of the molecular beacon, and a quencher is attached to the other end. When the probe is unbound, hairpin formation occurs, bringing the reporter and quencher into proximity and fluorescence is quenched.
If a target sequence is present during the annealing step of an amplification reaction, the loop portion of the molecular beacon binds to its target sequence, causing the stem to denature. The reporter and quencher are thus separated, quenching is diminished, and the reporter fluorescence is detectable. Because fluorescence is emitted from the probe only when it is bound to the target, the amount of fluorescence detected is proportional to the amount of target in the reaction. Again, a properly designed molecular beacon can be used to distinguish underlying sequence variations, i.e. genotypes, within the amplified sequence. Typically, this is accomplished with melting curve analysis following PCR.
Dual Hybridization Probes
These assays use two sequence-specific oligonucleotide probes which bind to adjacent sequences in the target. The probes are labeled with a pair of dyes that can engage in fluorescence resonance energy transfer (FRET). The donor dye is attached to the 3′ end of the first probe, while the acceptor dye is attached to the 5′ end of the second probe. This order may be reversed, so long as binding of both oligonucleotides to the target brings the fluorophores within FRET range (Forster radius).
During real-time PCR, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. At the annealing step, the probes hybridize to their target sequences in a head-to-tail arrangement. This brings the donor and acceptor dyes into proximity, allowing FRET to occur. The amount of acceptor fluorescence is proportional to the amount of PCR product present. Hybridization probes enable a variety of genetic detection and quantification readouts.
Primer/Probe Combinations
These detectors use a sequence specific oligonucleotide primer and a sequence specific oligonucleotide probe. The primer and the probe are designed to bind to adjacent sequences of the target, usually with the probe complementary to the strand formed by the primer. The probe and the primer are labeled with a pair of dyes that can engage in (FRET). Generally, the donor dye is attached near the 3′ end of the primer, while the acceptor dye is attached to the 3′ end of the probe, which anneals to the complementary strand synthesized by primer extension.
As with the dual hybridization probes, during DNA amplification, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. At the annealing step, the probe and primer hybridize to their target sequences in a head-to-tail arrangement. This brings the donor and acceptor dyes into proximity, allowing FRET to occur. The increasing amount of acceptor fluorescence is proportional to the amount of PCR product present.
Dynamic Flux Amplification
An amplification method described in the art comprises determining the melting temperature of the target sequence and setting the upper limit of the thermal cycle temperature to maximize the denaturation of the target sequence while minimizing the denaturation of the non-target sequences (dynamic flux amplification or DFA). This approach fosters the creation of a bubble as the reaction is heated to a temperature approaching the denaturation temperature of the target sequence. Assuming the denaturation temperature of the target sequence is less than the adjacent sequences, the adjacent sequences will remain annealed, resulting in a bubble forming in the DNA strand as the target sequence denatures. Of course, it is probable that multiple bubbles form at various points along the DNA sequence that possess a similar denature temperature to the target sequence. Nevertheless, the total amount of un-denatured sequence is still less than would be the case if the upper temperature was raised to 95° C. or more.
One advantage of controlling the denaturation temperature to create a nucleic acid bubble is that it significantly limits the formation of nonspecific product by preventing the binding of the primers to sites other than the target sequence, by making such sites unavailable for hybridization. This results from the target sequence being favored to denature relative to non-target regions of the target genome and thereby significantly reduces the available sequence that can serve as non-specific binding sites during the amplification process.
One disadvantage of the aforementioned conventional probe chemistries is that they are not compatible with Dynamic Flux Amplification (“DFA”) technology. This is due in part to the difference in required melting temperatures of the probes used in PCR as compared to DFA. PCR utilizes probes that are generally in the 20-30 base pair range and generally possess a Tm of at least 20° C. less than the Tm of the sequence of interest. In contrast, DFA requires probes that are within 20° C. or less of the Tm of the sequence of interest. Because DFA normally operates outside of annealing temperature ranges used in probe technology for PCR, such probes as currently practiced are generally not compatible with DFA technology.
It would be desirable if existing PCR primers could be modified to take advantage of the narrow temperature range used in DFA or at the very least a thermal cycling range that is narrower than those used in conventional PCR and thus obviate the need to completely redesign primers in order to obtain an increase in speed. The narrow temperature range can be used as a target temperature range in order to identify, design and/or generate specific primers that have sufficiently high Tm values when hybridized with the target nucleic acid.
It would be desirable to have an amplification method that significantly eliminated the formation of undesirable product by inhibiting the extension of the reaction beyond the amplification bubble.
Often the primers with the necessary Tm ranges must be designed de novo. Thus, although users of traditional PCR assays may desire increased speed, the cost of designing, evaluating and optimizing the primers for DFA necessary to obtain the narrower cycling range is frequently prohibitive, locking users into the slower conventional PCR, rather than taking advantage of the increased speed possible from dynamic flux amplification.
Thus, there is a need in the art to develop primers and probes, other reagents, and methodologies, which are compatible with DFA. Specifically, there is an unmet need in the art to develop primers and probes that can be utilized in DFA protocols.
In some aspects, the term “extreme chain reaction” or “XCR” will be utilized in the description. The present inventors utilize the term XCR as a synonym for DFA. Thus, the two terms are used interchangeably.
Multiplex Detection
The need for, at a minimum, the ability to detect two or more distinct amplified targets within a single reaction is a fundamental aspect of modern diagnostic tests. Although some tests can be brought to market with separate reaction vessels containing the necessary test performance controls, it is cost effective in terms of sample throughput, and reagent usage, to incorporate the reaction controls within a single reaction vessel. Effective utilization of DFA ideally would involve a means to detect one or more amplified targets simultaneously.
Another consequence of being able to custom design target denaturation and primer annealing temperatures while simultaneously narrowing the thermal cycling range allows for amplification of different targets to be carried out in a single reaction vessel by thermal cycling the reaction vessel at different temperature ranges in succession.
Probe technology for use with both PCR primers as well as the high Tm and frequently longer primers commonly used in DFA have been disclosed in WO 2015/054516 (incorporated herein in its entirety for all purposes).